Superoxide dismutase (SOD): SOD catalyzes the dismutation of O₂ ^−. to H₂O₂ (Fig. 1.2). There are three isoforms of SOD in mammalian cells including SOD1 (CuZnSOD), SOD2 (MnSOD), and SOD3 (extracellular SOD). Of the three isoforms, SOD2 is exclusively expressed in the mitochondrial matrix [27]. In contrast to SOD1 and SOD2, genetic knockdown of SOD2 causes early neonatal death in mice [28] and endothelial deficiency and dysfunction in apolipoprotein E (ApoE)-deficient mice [29].

Catalase: The enzyme catalase, which is primarily localized in peroxisomes, catalyzes the dismutation of H₂O₂ to water and O₂ (Fig. 1.2). At least three types of catalases have been described, which include the classic Fe heme enzymes, manganese (Mn) enzymes, and the catalase-peroxidases [30]. Although catalase is a key enzyme involved in dismutation of mitochondrial and non-mitochondrial H₂O₂, knockdown of catalase in mice has no detrimental developmental effects; however, they showed differential sensitivity to oxidant injury. It therefore appears that catalase is dispensable and other detoxifying pathways can compensate [31].

Glutathione peroxidase: Glutathione peroxidase (GPx) reduces lipid hydroperoxides to their corresponding alcohols and further to H₂O₂ to water (Fig. 1.2). GPx1-4 is a family of isoenzymes homologous to the selenocysteine GPx1 and use reduced glutathione (GSH) as a co-substrate in the reduction of lipid hydroperoxides or H₂O_2. Not all GPxs use GSH nor do they contain selenocysteine at the active site; however, these are thioredoxin-dependent peroxidases containing a redox-sensitive cysteine or a selenocysteine. GPx1 is the most abundant GPx isoform present in all cells and primarily localized in the cytosol, mitochondria, and in peroxisomes of some cells [32, 33]. In addition to reducing H₂O₂ and lipid hydroperoxides, GPx1 may also act as a peroxynitrite reductase [34]. However, the in vivo role of GPx1 in modulating peroxynitrite levels is unclear, but lack of GPx1 enhances survival to peroxynitrite [35] through an as yet undefined mechanism. Knockdown of GPx4, but not GPx1, is embryonically lethal [36]; however, GPx1 knockout mice exhibit susceptibility to ischemia-reperfusion injury in mouse myocardium [37]. The effect of knockdown of GPX1 in mice on mitochondrial ROS production is, however, unclear.

Thioredoxin: Thioredoxins (Trxs), with a dithiol/disulfide active site (CGPC), are the major cellular protein disulfide reductases [38], and serve as electron donors for enzymes such as ribonucleotide reductases, thioredoxin peroxidases (peroxiredoxins), and methionine sulfoxide reductases [39]. Trx isoforms are present in most organisms; trx1 localizes within the cytosol and translocates to the nucleus during oxidative stress, whereas trx2 is exclusively located in the mitochondria [40]. Trxs are critical for redox regulation of protein function and signaling via thiol redox control. A growing number of transcription factors including NF-kB or the Ref-1-dependent AP1 require Trx reduction for DNA binding. The cytosolic mammalian Trx, lack of which is embryonically lethal, has numerous functions such as defense against oxidative stress, control of growth, and apoptosis. It is also secreted and has co-cytokine and chemokine activities. Trx reductases of higher eukaryotes are larger (112–130 kDa), selenium-dependent dimeric flavoproteins with a broad substrate specificity that also reduce nondisulfide substrates such as hydroperoxides, vitamin C, or selenite. Mammalian mitochondrial Trx is a monomer of approximately 12 kDa, while mammalian mitochondrial Trx reductase is a selenoprotein homodimer of a 55 kDa subunit. A function of the mitochondrial Trx system is as an enzyme that detoxifies the hydrogen peroxide generated by the mitochondrial metabolism. It is the electron donor for mitochondrial peroxiredoxin. Trx2 deficiency is embryonically lethal at E10.5 d, which coincides with the maturation of mitochondrial function.

Peroxiredoxin: Peroxiredoxins (Prxs) are a family of six small non-seleno thiol-specific peroxidases and are important regulators of cellular ROS. Prx III is localized in the mitochondria, while Prx V is located both in the mitochondria and peroxisomes [41]. All Prxs reduce H₂O₂ and organic hydroperoxides to H₂O and alcohol, respectively, using reducing equivalents from thiol-containing proteins such as Trxs [42]. All the three systems, GPX/GR, Trx/TrxR, and Prx, rely on NADPH as a source of reducing equivalents and mitochondrial NADPH can be regenerated by NADH-supported reduction of NADP+ via energy-dependent trans-hydrogenation. In the mitochondria, the NADPH reduction is carried out by three mitochondrial enzymes: isocitrate dehydrogenase, malic enzyme, and transhydrogenase [43]. As mitochondrial pools of NADPH and GSH are rather large [44, 45], prolonged ROS generation will depend on the antioxidant defense systems that require regeneration of NADPH and GSH. In mice, overexpression of mitochondrial Prx III protected against left ventricular remodeling and myocardial infarction [46]. Thus, mitochondrial redox homeostasis is achieved through cooperation between GSH/GSSG redox coupling in conjunction with redox proteins, GPx, Trx2, and Prx III.

Nuclear factor erythroid 2-related factor 2: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that is activated in response to oxidative stress and upregulates antioxidant and phase II detoxification enzymes. Nrf2 is activated by oxidizing extracellular conditions that leads to enhanced mitochondrial ROS production, dissociation of Nrf2 from Keap1, and translocation of Nrf2 to the nucleus, binding to the ARE promoter sequences of cytoprotective genes resulting in induction and expression of antioxidant and anti-inflammatory proteins. A potential link between Nrf2, Trx2, and mitochondrial ROS has been recently described [47]. Nrf2 activation can be inhibited by Trx2 overexpression resulting in decreased mitochondrial ROS [48]. Further support comes from decrease in the expression of genes regulated by Nrf2 [49]. Thus, it appears that extracellular redox states mediate Nrf2 signal transduction through ROS generated in the mitochondria and may be regulated by relative concentrations of Trx2. It is noteworthy that other signal transduction pathways, such as TNFα signaling, are also reliant on mitochondrial ROS production, and these studies have shown Nrf2/Trx2 to function as a modulator of signal transduction [50]. Since ROS production is increased as a consequence of oxidizing extracellular conditions and the inhibition of ROS generation decreases Nrf2 activation, it is therefore possible that ROS may be directly responsible for modification of Keap1 enroute to the activation of Nrf2. However, it is currently unknown whether mitochondrial ROS act directly on the Keap1/Nrf2 system or whether mitochondrial ROS affect other regulatory machinery that may indirectly affect signal transduction through (de)activation of other redox-sensitive components. Since oxidant stress conditions are evident in the pathogeneses of many respiratory diseases, furthering our understanding on the mechanistic control of extracellular-induced Nrf2 activation may lead to the development of potential therapeutic interventions. A detailed description of regulation of mitochondrial function by Nrf2 is provided in the accompanying chapter.

Sirtuins: Sirtuins, NAD+-dependent histone/protein deacetylases, catalyze the hydrolysis of acetyl groups from the side chain amino group of lysine residues in proteins. The deacetylation requires NAD+ and generates nicotinamide and 2′-O-acetyl-ADP-ribose [51]. Seven sirtuins members have been identified in mammals, SIRT1–7; SIRT3, 4, and 5 are primarily located in the mitochondria [52]. SIRT1, 6, and 7 are found in the nucleus, whereas SIRT2 is cytosolic, with robust deacetylase activity. There is strong evidence that supports a role for SIRT1 in mounting a response to oxidative stress by directly deacetylating several transcription factors that regulate expression of antioxidant genes. Notably, SIRT1 activates several members of the FOXO family of transcription factors which promote the expression of stress response genes including SOD2 [53–55]. SIRT1 also promotes mitochondrial biogenesis by activating peroxisome proliferator-activated receptor co-activator 1-α (PGC-1α) [56], which increases mitochondrial mass and upregulates the expression of oxidative stress genes including GPx1, catalase, and MnSOD [57]. Also, SIRT1 inactivates the p65 subunit of NF-ĸB through direct deacetylation. NF-ĸB inhibition suppresses the inducible iNOS and nitrous oxide production and thus may lower the cellular ROS load [58]. Mitochondrial SIRT3 deacetylates and activates several enzymes that are critical in maintaining mitochondrial and cellular ROS levels. SIRT3 deacetylates SOD2 at two important lysine residues to boost its catalytic activity, and the deletion of S1RT3 results in loss of catalytic activity of SOD2 [59]. There is evidence that SIRT3 plays an important ameliorative role in preventing the pathological response to pressure overload and aging-associated decline in cardiac function [60]; however, the role of SIRT3 in mitochondrial ROS-dependent respiratory diseases is yet to be defined.

Mitochondria-targeted antioxidant therapies: Given the central role of mitochondria and mitochondrial ROS in human pathologies, several natural antioxidants such as vitamin C, vitamin E, rottlerin, curcumin, ginsenoside Rb1, and epoetin delta have been investigated both in vitro and in vivo for their antioxidant efficacy, and most of these were not found to be effective in attenuating mitochondrial ROS production in response to an environmental stimulus [61]. In the last two decades, there has been considerable advancement in the development of mitochondria-targeted small molecule antioxidants that exhibited reduced mitochondrial oxidative damage and partially prevented decline in mitochondrial function in some of the pathologies. Several of the small molecule antioxidants such as alpha-tocopherol, ubiquinone, piperidine nitroxide, and CP have been conjugated to the lipophilic cation, triphenylphosphonium (TPP), to generate mitochondria-targeted small molecule antioxidants Mito-E2, Mito-Q, Mito-CP, and Mito-TEMPOL, respectively (Fig. 1.3).

Acute lung injury: Acute lung injury (due to sepsis or ventilator-induced lung injury) and subacute lung injury (due to ionizing radiation-induced lung injury) share profound increases in vascular permeability as a key element driving increased morbidity and mortality. There is evidence demonstrating a potential link between sepsis and mitochondrial dysfunction both in septic patients and animal models of sepsis. Ultrastructural abnormalities in mitochondrial morphology including irregular cristae were reported from liver and skeletal muscle biopsies obtained from septic patients who died in ICU [87]. Similarly, in animal models of sepsis, significant alterations of mitochondrial ultrastructure and abnormalities have been described [88–91]. One postulated mechanism is changes in mitochondrial function due to inhibition of mitochondrial respiratory chain, which may contribute to a decrease in oxygen utilization in acute lung injury [92]. Recent studies have identified the involvement of mediators such as TNF-α, ROS, NO, and peroxynitrite in the inhibition of mitochondrial dysfunction [93]. TNF-α, induced in macrophages and lymphocytes by endotoxin, plays a major role in sepsis-mediated lung inflammation and injury via increase in mitochondrial Ca²⁺ and ROS production and signaling [94, 95]. In severe sepsis, there is evidence for a massive influx of extracellular Ca²⁺ via Ca²⁺ release-activated (CRAC) channel or by opening of voltage-, receptor-, or IP₃-operated channels, which is partly the case in mitochondria leading to swelling, mitochondrial dysfunction, and ultimately cell death. While therapies for sepsis are limited, a substantial body of evidence from animal models suggests a beneficial role for mitochondrial targeting with antioxidants such as Mito-Q against endotoxin-mediated sepsis [66]. Further, mitochondria-targeted antioxidants reduced IL-6 release and oxidative stress and improved mitochondrial function in a rat model of acute sepsis [67]. These studies suggest that antioxidants targeted to mitochondria may be an important therapeutic approach in the management of sepsis and multiple organ dysfunction syndrome. A recent study on soluble TNF-α-mediated shedding of the TNF-α receptor 1 ectodomain via increased mitochondrial Ca²⁺ and ROS and TNF-α-converting enzyme may limit inflammatory response in sepsis [94]. The importance of mitochondrial dysfunction in sepsis was also demonstrated in studies related to administration of bone-marrow-derived stromal cells (BMSCs). Exogenous administration of BMSCs offered protection in mouse models of sepsis-induced acute lung injury [96, 97]. The protection was attributed to Cx43-dependent alveolar attachment and transfer of mitochondria from BMSCs to alveolar epithelial cells [98]. Future studies on mechanisms regulating mitochondrial function may lead to targeted therapeutic approaches for treating sepsis-induced pulmonary inflammation and injury.

Allergic bronchial asthma: Asthma is a complex lung disease characterized by airflow obstruction, airway hyperresponsiveness, and airway inflammation. Exposure to allergens and environmental pollutants induces innate and acquired immune systems to release proinflammatory and bioactive mediators which lead to recruitment and activation of various inflammatory cells such as eosinophils, mast cells, macrophages, neutrophils, lymphocytes, and platelets [99]. Recent studies have revealed the involvement of mitochondria and mitochondrial dysfunction in asthma pathogenesis. Mitochondrial DNA changes and mutations play a role in the development of asthma. Polymorphisms or haplotype differences in the mitochondrial genome may influence the severity of asthma in humans [100, 101]. Human mitochondrial DNA defects have been observed to accumulate with age and in age-related neurodegenerative diseases such as Alzheimer’s and Parkinson’s, which are induced by free radicals and ROS. Although no mitochondrial DNA changes are known to date, age-dependent mitochondrial defects may play a critical role in determining the susceptibility to asthma in the elderly. In addition to mitochondrial DNA, mitochondrial tRNA mutations along with specific rRNA mutations have been significantly more frequent in asthmatic patients compared to control subjects suggesting a role for mitochondrial genetic background in asthma pathogenesis [102]. Involvement of mitochondrial ROS in the pathogenesis of bronchial asthma has received considerable attention in recent years. Several lines of evidence in animal models of asthma and human asthma suggest that ROS released by activated macrophages and granulocytes induces oxidative damage in mitochondria of airway epithelial cells, thereby increasing mucus secretion and epithelial permeability [103–105]. Inhalation of allergens and environmental pollutants induces airway inflammation, releasing proinflammatory mediators such as histamine, PGE2, leukotrienes, and NO. These mediators enhance airway smooth muscle contraction as well as recruit and activate various inflammatory cells into the alveolar space [99], which release ROS, peroxynitrite, and lipid peroxidation products, and they further damage the epithelium. Moreover, the asthmatic epithelium is more susceptible to apoptosis mediated by ROS, probably due to diminished antioxidant levels and decreased endogenous antioxidant enzymes in the peripheral tissues of asthmatic patients [105–107]. Finally, increased numbers of mitochondria and changes in mitochondrial ultrastructure and mitochondrial swelling have been shown in the bronchial epithelium of asthmatic mouse models and human asthmatics [108–110]. Mechanisms underlying increased mitochondrial ROS production are unclear; however, reduced expression of glucocorticoid and estrogen receptors in mitochondria of lung epithelial cells in human asthmatics may be involved in reduction in OXPHOS enzyme biosynthesis, mitochondrial impairment, elevated ROS production, and induction of apoptosis in epithelial and other cell types [10, 111, 112]. Additionally, glucocorticoid and estrogen receptors are involved in regulation of apoptotic, and inflammatory responses [113, 114] and reduced expression of both these receptors could contribute to reduced protection against apoptosis and inflammation in asthma [115]. Interestingly, preexisting mitochondrial dysfunction induced by environmental pollutants intensifies allergic airway inflammation implying that mitochondrial defects could be a risk factor for the development of allergic disorders in susceptible individuals [116]. Although no specific therapy is currently available to minimize mitochondrial dysfunction in experimental models and human asthma, mitochondria-targeted antioxidants may be a promising approach; however, they have yet to be tested as anti-asthmatic drugs.

Pulmonary arterial hypertension: Pulmonary arterial hypertension (PAH) is a lethal disease of the pulmonary vasculature characterized by pulmonary vasoconstriction, right ventricular hypertrophy, and right ventricular failure [117–119]. The hallmark of PAH is excessive proliferation of pulmonary artery smooth muscle cells that thicken the lumen and increase resistance in pulmonary arteries [120]. PAH is a complex vascular disease, and several environmental and genetic factors including hypoxia, loss-of-function mutations of bone morphogenetic protein receptor II, and viral infections have been linked to the development of PAH [121]; however, the cause and mechanisms of the vascular remodeling in PAH remain undefined. Abnormal mitochondria in pulmonary artery smooth muscle cells may suppress mitochondria-dependent apoptosis and contribute to the vascular remodeling in PAH and recent studies indicate a role for Nogo-B in hypoxia-mediated disruption of mitochondria-endoplasmic reticulum stress in mice and human PAH [122]. Several studies have implicated a role for ROS and RNS derived from Nox proteins and mitochondria in the development of PAH [118]. ROS derived from mitochondria may contribute to mitochondrial dysfunction through two pathways. In the first pathway, excess mitochondrial ROS derived from mitochondrial electron transport chain may promote cellular senescence, necrosis, or apoptosis, leading to endothelial dysfunction and vasculopathy [123]. In the second pathway, mitochondrial dysfunction a reduction in H₂O₂ levels may trigger the pathogenesis of PAH. According to this model, changes in mitochondria result in decreased H₂O₂ production, leading to decreased cellular redox potential causing cellular depolarization, opening of voltage-gated potassium Kv1.512, influx of Ca²⁺, and vasoconstriction of pulmonary artery SMCs [124]. Several studies also have indicated impaired NO signaling and bioavailability in patients with PAH and in animal models of PAH [125–127]. Although mitochondrial dysfunction and mitochondrial ROS and RNS are implicated in PAH, potential antioxidant strategies specifically targeting mitochondrial ROS as therapy in PAH have been investigated only in animal models, and they need to be extended to humans.

Pulmonary fibrosis: Pulmonary fibrosis is a lung disease characterized by irreversible destruction of lung architecture, abnormal wound healing, and deposition of extracellular matrix proteins leading to organ dysfunction, disruption of gas exchange, and death from respiratory failure. Lung fibrosis could be idiopathic [128] or arise from exposure to environmental toxins such as fibers, asbestos, metals, pesticides, chemotherapeutic drugs, viruses, multiwalled carbon nanotubes, and radiotherapy [129, 130]. The underlying mechanisms of idiopathic pulmonary fibrosis (IPF) or environmental agents mediated lung fibrosis are poorly understood. However, at the cellular level, injury to the bronchial and alveolar epithelium, epithelial-mesenchymal transition (EMT), activation of macrophages and proliferation, and transformation of fibroblasts to myofibroblasts may all contribute to pulmonary fibrogenesis [131]. At the molecular level, the fibrogenic responses have been linked to growth factors, ROS, TGF-β, matrix metalloproteinases, bioactive lipids such as sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), and its G-protein-coupled receptors acting via specific signaling pathways that modulate fibrogenesis in the lung. There is compelling evidence for the role for ROS, TGF-β, TGF-β-mediated ROS, and ROS-dependent TGF-β activation in the development of pulmonary fibrosis in animal models as well as IPF [132–134]. In animal models of asbestos and bleomycin-induced fibrosis and IPF lungs, there is compelling evidence for oxidative stress with increased production of ROS, which could modify macromolecules and alter cellular functions of epithelial cells and fibroblasts [135]. ROS derived from NOX1, NOX2, and NOX4 has been implicated in the pathogenesis of pulmonary fibrosis [136, 137]; however, recent studies with asbestos fibers suggest that activation of mitochondrial ROS and the resultant damage on lung tissue, as observed in a murine model of asbestosis-induced lung fibrosis, are important [138]. Further, macrophage-derived mitochondrial H₂O₂ seems to play a key role in asbestos fiber-mediated pulmonary fibrosis [138, 139] that was blocked by catalase or knockdown of iron-sulfur protein of complex III in the mitochondrial electron transport chain, a major site of ROS production [140]. Further, accumulating data suggest a potential role for epithelial endoplasmic reticulum stress and mitochondrial ROS in alveolar epithelial cell apoptosis in IPF and asbestos-mediated pulmonary fibrosis [141, 142] (Fig. 1.4). In addition to ROS, dysregulation of TGF-β expression and/or signaling was shown to play an important role in the pathogenesis of pulmonary fibrosis. Interestingly, the interactions between ROS and TGF-β influence the process of fibrogenesis. ROS, derived from NOX and mitochondria, activates latent TGF-β and induces TGF-β expression in many cell types including alveolar epithelial cells and macrophages [143], and conversely, TGF-β also increases ROS production by activating NOX4 [136, 137] and activation of complex III of the mitochondrial electron chain [144]. Mitochondrially targeted antioxidants or genetic disruption of mitochondrial complex III significantly attenuated TGF-β-induced profibrotic gene expression suggesting that targeting mitochondrial ROS may be beneficial in pulmonary fibrosis [144].

Bronchopulmonary dysplasia: Bronchopulmonary dysplasia (BPD) is one of the devastating complications of premature newborns with no proven therapy. The development of BPD is associated with a number of risk factors including hyperoxia, ventilator-induced lung injury, and pre- and postnatal infections [145]. An arrest of alveolar development, secondary to mitochondrial bioenergetics failure and reduced ATP production, is one of the hallmarks of BPD [146]. Supplemental oxygen therapy (ventilator) resulting in oxygen toxicity, mediated by production and/or accumulation of ROS, has been implicated in the development of BPD in premature infants and experimental models of hyperoxia-induced injury [147, 148]. Hyperoxia generates excessive ROS, which can directly target and injure alveolar epithelium and lung endothelium; however, the source of ROS in hyperoxia-induced lung injury remains controversial. Some of the earlier reports suggest that mitochondria-derived ROS is a critical determinant of hyperoxic cell damage and apoptosis using respiration-deficient cell lines [149, 150]. Additionally, using nine genetically engineered mice deficient in or overexpression of proteins, a role for mitochondrial oxidants initiating BAX- or BAK-dependent alveolar epithelial cell death contributing to hyperoxia-induced lung injury has been demonstrated [151]. This is in contradiction to an earlier report that showed that hyperoxia-induced cell death to be independent of mitochondrial ROS but requires expression of Bax or Bak proteins [152]. Further, hyperoxia increased ROS formation in pulmonary capillary endothelial cells of rat lungs, which was blocked by complex I inhibitor rotenone as well as inhibitors of NADPH oxidase and the intracellular calcium chelator BAPTA [153]. These data suggest a role for both mitochondrial and NOX-derived ROS from the pulmonary endothelium in situ in hyperoxia-induced lung injury. However, exposure of human lung endothelial cells to hyperoxia enhanced ROS generation that was dependent on NOX activation and independent of mitochondrial electron transport chain [154]. There is no clear explanation for the varying observations on the involvement of mitochondrial vs. NOX proteins in hyperoxia-induced ROS generation; however, use of different cell types, variation due to the use of mouse, rat, and humans, or differential levels of antioxidants in various cell types may all account for the discrepancies. Studies in animal models and premature infants have shown that expression and activity of antioxidant enzymes (AOEs) increase during the third trimester in utero, and premature infants have relative low levels of AOEs [155] that can upset redox balance of the cell leading to apoptosis and inhibition of lung development [151, 156]. Animal studies point out that adult animals are more vulnerable to hyperoxia compared to neonates [157, 158] suggesting reduced cellular antioxidant capacities relative to the newborn animals. Although developmental differences in oxidative stress responses were observed in the murine lung, greater lethality of adult animals exposed to hyperoxia may be due to inflammation rather than to differences in AOEs [159]. The mechanism(s) of increased ROS generation by changes in oxygen pressure from 150 to 300 mm of mercury is unclear, but hyperoxia stimulates signaling pathways mediated by Rac1 [153, 160], MAP kinases [154, 161], PI3 kinase-Akt [162], cytoskeletal redistribution [163], and formation of lipid rafts [164] that contribute to NOX-dependent and NOX-independent ROS formation ultimately leading to lung injury. A recent study suggests the potential involvement of sphingosine-1-phosphate (S1P) and sphingosine kinase (SphK) 1 signaling axis in a murine model of neonatal BPD [165]. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues and Sphk1 ^−/− , but not Sphk2 ^−/− or Sgpl1 ^+/− , mice offered protection against hyperoxia-induced lung injury with improved alveolarization and alveolar integrity compared to their wild-type counterparts. Further, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX 4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells (HLMVECs) showed that exogenous S1P stimulated intracellular ROS generation, while transfection with SphK1 siRNA or the use of specific SphK inhibitor attenuated hyperoxia-induced S1P generation. Further, knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of BPD [165]. Further studies are necessary to delineate the therapeutic potential of small molecule inhibitors of sphingosine kinase 1 as well as other enzymes involved in S1P metabolism and antioxidant therapy such as thioredoxin treatment [166] on hyperoxia-mediated alveolar simplification, cell death, and mitochondrial dysfunction.